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Loss of centromeric histone H3 (CENH3) from

centromeres precedes uniparental chromosome


elimination in interspecic barley hybrids
Maryam Saneia, Richard Pickeringb,1, Katrin Kumkea, Shuhei Nasudac, and Andreas Houbena,2
a
Leibniz Institute of Plant Genetics and Crop Plant Research, 06466 Gatersleben, Germany; bNew Zealand Institute for Plant and Food Research Limited,
Private Bag 4704, Christchurch, New Zealand; and cLaboratory of Plant Genetics, Graduate School of Agriculture, Kyoto University, 606-8502 Kyoto, Japan

Edited by Hugo K. Dooner, Waksman Institute, Rutgers University, Piscataway, NJ, and approved June 14, 2011 (received for review February 28, 2011)

Uniparental chromosome elimination occurs in several interspecic


hybrids of plants. We studied the mechanism underlying selective
elimination of the paternal chromosomes during the early development of Hordeum vulgare Hordeum bulbosum embryos. The
following conclusions regarding the role of the centromere-specic
histone H3 variant (CENH3) in the process of chromosome elimination were drawn: (i) centromere inactivity of H. bulbosum chromosomes triggers the mitosis-dependent process of uniparental
chromosome elimination in unstable H. vulgare H. bulbosum
hybrids; (ii) centromeric loss of CENH3 protein rather than uniparental silencing of CENH3 genes causes centromere inactivity; (iii) in
stable species combinations, cross-species incorporation of CENH3
occurs despite centromere-sequence differences, and not all CENH3
variants get incorporated into centromeres if multiple CENH3s are
present in species combinations; and (iv) diploid barley species encode two CENH3 variants, the proteins of which are intermingled
within centromeres throughout mitosis and meiosis.
kinetochore

| interspecies hybridization | micronuclei | wide crosses

hromosome elimination of one parental genome after fertilization of the egg by the sperm of another species is a fairly
common phenomenon and results in the formation of haploid
embryos. It has been exploited for barley (1) and other species
(e.g., wheat, potato) to produce doubled haploids for breeding
and mapping purposes (reviewed in 2). The advantage of doubled haploids for breeders is that homozygosity can be achieved
in the rst generation, whereas in breeding systems, such as
pedigree or backcrossing, several selfed generations are needed
to obtain high levels of homozygosity.
To produce haploids of barley, crosses are made with Hordeum bulbosum Linnaeus (bulbous barley grass), a close relative
of cultivated barley in the secondary gene pool. Chromosomes of
H. bulbosum are eliminated several days after pollination (1, 36)
independent of the crossing direction (1), but hybrids combining
both sets of parental chromosomes can be obtained (7). Chromosome elimination is known to depend on genetic factors (7)
and temperature after fertilization (8).
Several explanations have been proposed to account for uniparental chromosome elimination [e.g., difference in timing of
essential mitotic processes attributable to asynchronous cell cycling (9), asynchrony in nucleoprotein synthesis leading to a loss
of the most retarded chromosomes (3, 10)]. Other hypotheses
that have been put forward are the formation of multipolar
spindles (5), spatial separation of genomes during interphase
(11, 12), and genome elimination by nuclear extrusions (4, 13).
In addition, degradation of alien chromosomes by host-specic
nucleases (14), uniparental nondisjunction of anaphase chromosomes (15), and parent-specic inactivation of centromeres
(11, 1619) have been suggested. The actual cellular mechanism
involved in the process of uniparental chromosome elimination
remains poorly understood.
To test whether parent-specic inactivation of centromeres
is involved in the mitosis-dependent process of chromosome
E498E505 | PNAS | August 16, 2011 | vol. 108 | no. 33

elimination in interpecic hybrids, we analyzed the centromerespecic histone H3 variant (CENH3) [originally called CENP-A
in humans (20) and HTR12 in Arabidopsis thaliana (21)] in
chromosomally unstable and stable Hordeum vulgare H. bulbosum combinations. CENH3 was selected for our study because
in mammals (22), Caenorhabditis elegans (23), and Drosophila
melanogaster (24), its loss results in the failure of centromere
formation and chromosome segregation. A region in CENH3
dened as the centromere targeting domain (CATD) is critical
for centromeric localization of CENH3 in various species (25
27). The CATD is composed of the loop1 linker and 2-helix of
CENH3 (28, 29), and its substitution enabled the incorporation
of an H3 chimera into centromeres (26). This domain mediates
molecular recognition events before and after nucleosome assembly and is important for binding of CENH3 to centromeric
DNA (27, 28, 30), to CENH3-specic chaperones (3133), and
to CENH3-stabilizing factors (34, 35).
Although centromeric DNA sequences are extremely diverse,
all eukaryotic centromeres contain CENH3 (36). The chromosomal location of CENH3 is the assembly site for the kinetochore complex of active centromeres. Any error in histone gene
transcription, translation, modication, or import could affect
the ability to assemble intact CENH3 chromatin, which would
result in the loss of CENH3 from centromeres and, hence, of
centromere identity (reviewed in 37). In contrast to conventional
histones, CENH3 is rapidly evolving and shows signatures of
adaptive evolution in some species (38). ChIP data indicated
that CENH3 interacts with H. vulgare with CEREBA, a centromeric retroelement-like element conserved among cereal centromeres, and with H. bulbosum- and H. vulgare-specic GC-rich
centromeric satellite sequences (39).
The present work provides insights into the role of CENH3 in
the process of selective elimination of paternal chromosomes
during the development of H. vulgare H. bulbosum hybrid
embryos. In addition, we identied two functional CENH3s in
both diploid barley species and assayed their incorporation into
centromeres of alien chromosomes. We found that despite the
presence of transcripts, not all parental CENH3 variants are
incorporated into centromeres if multiple CENH3s coexist in

Author contributions: A.H. designed research; M.S., R.P., K.K., and S.N. performed research; R.P. and S.N. contributed new reagents/analytic tools; M.S. analyzed data; and
M.S. and A.H. wrote the paper.
The authors declare no conict of interest.
This article is a PNAS Direct Submission.
Data deposition: The sequences reported in this paper have been deposited in the
GeneBank data base (accession nos. JF419328, JF419329, GU245882.1, and JF419330).
See Commentary on page 13361.
1

R.P. has been retired since 2010.

To whom correspondence should be addressed. E-mail: houben@ipk-gatersleben.de.

See Author Summary on page 13373.


This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1103190108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1103190108

PNAS PLUS

species combinations. Thus, the lack of cross-species CENH3


incorporation might act as a barrier to species hybridization.
Results

Fig. 1. Anaphase chromosome segregation behavior of normally segregating (A) and lagging (B) H. bulbosum chromosomes in an unstable H.
vulgare H. bulbosum hybrid embryo. Chromosomes of H. bulbosum
(green) were identied by genomic in situ hybridization using labeled genomic DNA of H. bulbosum. Chromosomes of H. vulgare are shown in blue.
Note the lagging chromosomes of H. bulbosum in B. (Scale bar: 10 m.)

Sanei et al.

Fig. 2. Anaphase chromosomes of an unstable (A) and stable (B) H. vulgare


H. bulbosum hybrid embryo after immunostaining with anti-grass CENH3
and anti-tubulin. The centromeres of lagging chromosomes (arrowheads)
are CENH3-negative. (Scale bar: 10 m.)

centromeric FISH signal clusters overlapped with the position of


strong CENH3 signals (Fig. 3E), suggesting that interphase
centromeres of H. bulbosum also carry less CENH3 protein.
Thus, centromere inactivity of H. bulbosum chromosomes,
by means of a loss of CENH3, triggers the mitosis-dependent
process of uniparental chromosome elimination in unstable
H. vulgare H. bulbosum hybrids.
At the end of mitosis during reformation of nuclear membranes,
lagging chromosomes form micronuclei (42, 43). To determine the
centromeric and transcriptional activity of micronucleated H.
bulbosum chromatin (Fig. 4A), the presence of CENH3 and of the
RNA polymerase II was assayed. Most micronuclei were CENH3negative (Fig. 4B), and only occasionally were weak CENH3
immunosignals seen. RNA polymerase II signals were distributed
homogeneously throughout the nucleoplasm of normal nuclei
(Fig. 4C), whereas micronuclei were weakly immunolabeled (Fig.
4C). Because heterochromatinization of eliminated chromatin is
a well-known phenomenon in many nonplant organisms (44), we
tested next whether micronuclei are enriched in the heterochromatin marker histone H3 dimethylated at lysine 9 (H3K9me2).
Around 60% of 233 micronuclei analyzed revealed an enhanced
level of H3K9me2 compared with normal nuclei (Fig. 4D). Hence,
the transcriptional activity of micronucleated chromatin is strongly
reduced. Because the centromeres of micronucleated chromosomes are inactive, no further segregation of H. bulbosum chromosomes is possible after micronucleus formation.

Fig. 3. Interphase nucleus of an unstable H. vulgare H. bulbosum hybrid


embryo (A) after immunostaining with anti-grass CENH3 (B), genomic in situ
hybridization with H. bulbosum DNA (C, red), and in situ hybridization with
the Hordeum centromere-specic probe BAC7 (D). GISH, genomic in situ
hybridization. (E) Only approximately 7 of the 14 more or less equally sized
centromeric FISH signal clusters are overlapping with the position of strong
CENH3 signals. Hence, interphase centromeres of H. bulbosum carry less
CENH3 protein. BAC7-positive centromeres without CENH3-signals are shown
(arrows). (Scale bar: 10 m.)

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The fertilization of the H. vulgare egg by the H. bulbosum sperm is


followed by complete elimination of the H. bulbosum genome.
Depending on the genotype and on environmental conditions,
most of the H. bulbosum chromatin is eliminated after pollination
in almost all embryos within 1 wk (2). We rst analyzed the mitotic behavior of H. bulbosum chromosomes in dividing cells of
unstable hybrid embryos derived from H. vulgare H. bulbosum
(Cb2920/4). To promote chromosome elimination, a temperature
above 18 C was used for cultivation of pollinated H. vulgare
plants. In addition to H. bulbosum chromosomes with normal
mitotic movements (Fig. 1A), between 20% and 70% of anaphase
cells showed abnormally segregating H. bulbosum chromosomes
(Fig. 1B). Such chromosomes of H. bulbosum lagged behind H.
vulgare chromosomes, and the sister chromatids segregated
asymmetrically at anaphase. As previously described (4), the level
of mitotic chromosome condensation partly differed between the
parental genomes; chromosomes of H. bulbosum were often less
condensed. These observations are consistent with a loss of paternal chromosomes during cell division via lagging chromosomes
that later form micronuclei (3).
The centromere activity of hybrid cells undergoing uniparental
chromosome elimination was determined via immunostaining
with grass CENH3- and -tubulinspecic antibodies. Before
that, the cross-reactivity of the anti-grass CENH3 antibody used
(40) with CENH3 of H. bulbosum was conrmed (Fig. S1 A and
B). Fig. 2A shows typical results obtained in anaphase cells in
3- to 6-d-old unstable hybrid embryos. CENH3-positive active
centromeres were found in segregating chromatids, whereas
lagging chromosomes were depleted of CENH3 (Fig. 2A and Fig.
S2 AC). Neither a primary constriction nor a clear interaction
between -tubulin bers and kinetochores of lagging H. bulbosum chromosomes was recognizable. As a control, all centromeres of stable H. vulgare H. bulbosum (Cb3811/3) hybrids
cultivated below 18 C displayed CENH3 signals (Fig. 2B).
In addition, embryonic nuclei of unstable hybrids displayed
two classes of CENH3 signals. Approximately seven discrete
CENH3 signals of strong intensity, together with CENH3 signals
of less intensity, per nucleus were found (Fig. 3 B and E). Subsequent FISH with differentially labeled genomic DNA of H.
bulbosum (Fig. 3C) and with the Hordeum centromere-specic
probe BAC 7 (41) (Fig. 3D) conrmed the hybrid nature of those
nuclei. Only approximately 7 of the 14 more or less equally sized

SEE COMMENTARY

Uniparental Elimination of Chromosomes in Unstable H. vulgare H.


bulbosum Hybrids Is Accompanied by Loss of CENH3 from Centromeres.

CENH3s of Both Parental Genomes Are Transcribed in Chromosomally


Stable and Unstable Hybrid Embryos. To test whether H. bulbosum-

Fig. 4. Characterization of micronuclei of unstable H. vulgare H. bulbosum hybrid embryos. Micronuclei are H. bulbosum-positive after genomic in
situ hybridization (A) CENH3-negative (B) and RNA polymerase II-negative
(C) after immunostaining but enriched in H3K9me2-specic heterochromatin-specic markers (D). Arrowheads indicate micronuclei. (Scale bar: 10 m.)

specic CENH3 inactivation occurs, the transcriptional activity


of parental CENH3s in unstable hybrids was determined. Stable
and unstable hybrid embryos of different ages (57 d after pollination) were isolated, two to ve embryos of each age and type
were pooled, and RNA was isolated for cDNA synthesis. To
detect transcripts, we used CENH3 (primer pair 4/5) or
CENH3 (primer pair 6/7) type-specic primer pairs. H. vulgareand H. bulbosum-derived CENH3 transcripts were discriminated
via species-specic restriction sites. AlwI cleavage of the
H. bulbosum-derived PCR products generates two fragments of
210 bp and 54 bp, leaving H. vulgare-derived CENH3 fragments
undigested (Fig. S5A). Similarly, BanII cleavage of H. bulbosumderived CENH3 amplicons results in fragments of 398 bp and
234 bp, whereas H. vulgare-derived transcripts are unaffected
(Fig. S5B). BanII and AlwI cleavage of CENH3 transcripts amplied from stable or unstable H. vulgare H. bulbosum hybrid
revealed transcription of all four parental CENH3s (Fig. S5 A
and B). The expression patterns were similar regardless of the
embryo age. Thus, all CENH3 variants of both parental genomes
undergo transcription in unstable hybrids, and uniparental silencing of HbCENH3 genes is not the cause of chromosome
elimination in unstable hybrids.
Both CENH3 Variants Intermingle in Mitotic and Meiotic Centromeres
of H. vulgare. Duplication and maintenance of two or more copies

Both Hordeum Species Encode Two Active Variants of CENH3. Because silencing or biased expression of homologous genes has
been well documented in both natural and synthetic hybrids (45),
we rst isolated the corresponding genes with the aim of testing
whether the CENH3 gene of H. bulbosum undergoes silencing in
unstable hybrids.
Initial RT-PCR using a monocotyledon-specic CENH3 primer
pair (46) (Table S1, primer pair 1/2) for isolating on Hordeum
CENH3 amplied parts of the two CENH3 variants in H. vulgare
called HvCENH3 and HvCENH3. BLAST analysis against the
National Center for Biotechnology Information database resulted
in the identication of the 5 region of HvCENH3 (GenBank
accession no. BU996921). The coding sequence of HvCENH3
(GenBank accession no. AK249602) was completed by 3 RACE
PCR using primer 3 (Table S1). Based on the H. vulgare CENH3
sequences, primers were designed to amplify both CENH3s of H.
bulbosum, named HbCENH3 and HbCENH3 (Table S1, primer
pairs 14/15 and 1/2 for HbCENH3 and primer pair 6/7 and primer 3 for HbCENH3 were used for RACE PCR). The deduced
amino acid sequences of the identied CENH3s [GenBank accession nos. JF419328 (HvCENH3), JF419329 (HvCENH3),
GU245882.1 (HbCENH3), and JF419330 (HbCENH3)] were
compared with CENH3 of maize (47), rice (40), and sugar cane
(48) to determine the conserved N-helix, 1-helix, 2-helix,
3-helix, and CENH3-specic (26) loop1 regions (Fig. S3A).
Despite the rst ve completely conserved amino acids, the Nterminal tails were highly variable. CENH3 types have a shorter
N-terminal region than CENH3 types and a longer loop1 region.
Phylogenetic analysis of the amino acids of Hordeum CENH3s
with further plant CENH3s (Fig. S3B) showed that CENH3s
and CENH3s of Hordeum species form two distinct subclusters.
In addition, Hordeum CENH3s cluster with the CENH3s of
rice, maize, and sugar cane. Therefore, it is likely that and
types of CENH3 diverged before the speciation of H. vulgare and
H. bulbosum. Chromosome mapping of CENH3s conrmed this
assumption (Fig. S4). CENH3 is encoded by chromosome 1H in
H. vulgare and in H. bulbosum; CENH3 of rice maps to chromosome 5, which is syntenic to barley 1H (49). Furthermore,
CENH3 is located on chromosome 6H in both Hordeum species.
E500 | www.pnas.org/cgi/doi/10.1073/pnas.1103190108

of CENH3 seem to be rare events in both plant and animal


species. Examples of diploid plants that possess two CENH3s are
Arabidopsis halleri, Arabidopsis lyrata (50), and Luzula nivea (51).
Both variants are transcribed, but it is not clear whether they are
also functional. To characterize the chromosomal distribution of
multiple CENH3s in a diploid organism, antibodies were generated for each variant of H. vulgare CENH3. Western blot analysis
using in vitro translated HvCENH3 and HvCENH3 conrmed
the specicity of both antibodies (Fig. 5) because each type of
antibody recognized only the corresponding CENH3 variant,
whereas anti-grass CENH3 reacted with both types of CENH3.
To determine the chromosomal distribution of CENH3 and
CENH3 during mitosis and meiosis, immunouorescence ex-

Fig. 5. Western blot analysis demonstrating the specicity of antiHvCENH3-, anti-HvCENH3-, and anti-grass CENH3-specic antibodies on
nuclear (A) and in vitro translated HvCENH3 and HvCENH3 proteins (B).

Sanei et al.

periments were done. Double immunostaining revealed that


CENH3 and CENH3 are present in the centromeres of all
barley chromosomes at all mitotic stages analyzed (Fig. 6A and
Fig. S6A). Signals of both CENH3 variants always overlapped
with the position of the primary constriction. A similar distribution of both CENH3 variants was also detected at different
stages of the rst and second meiotic divisions (Fig. 6B and Fig.
S6B). The amount of CENH3 and CENH3 proteins seems to
be comparable, because the intensity of corresponding immunouorescence signals was generally of similar intensity.
To decipher the organization of CENH3- and CENH3containing centromeric chromatin at higher resolution, immunostaining experiments were done on extended chromatin bers.
For this purpose, chromatin bers prepared from isolated nuclei
of barley leaves were immunolabeled with both CENH3 antibodies. An overlap of CENH3- and CENH3-specic signals
was observed on centromeres (Fig. 6C) but also on up to 12-fold
articially extended centromeres (Fig. 6D). We conclude that
centromeres can incorporate CENH3 and CENH3 in all
chromosomes at mitotic and meiotic stages. Both CENH3 variants are intermingled in centromeric chromatin and are apparently equally involved in the formation of barley centromeres.
Although Transcribed, Not All CENH3 Variants Are Deposited on
Centromeres if Multiple CENH3s Coexist in Species Combinations. To

investigate whether both CENH3s of H. vulgare are incorporated


into centromeres of alien chromosomes, we studied stable H. vulgare H. bulbosum and Triticum aestivum H. vulgare combinations. First, we conrmed the species specicity of the barley
CENH3 antibodies. Both antibodies did not cross-react with H.
bulbosum or T. aestivum CENH3, whereas anti-grass CENH3
recognized the corresponding protein in all species tested (Fig.
S7). Next, double-immunostaining experiments were performed
on root nuclei of a stable H. vulgare H. bulbosum hybrid plant
with anti-grass CENH3 (as a positive control) combined with either HvCENH3- or HvCENH3-specic antibodies. Up to14
HvCENH3 and HvCENH3 signals overlapped with the position
Sanei et al.

PNAS PLUS
SEE COMMENTARY

Discussion
Diploid Hordeum Species Encode Two Functional CENH3 Variants.

This report describes two functional CENH3 variants in diploid


grasses. Because two CENH3 variants exist in H. vulgare and
H. bulbosum genes, duplication of CENH3 must have occurred at
least 7 million years ago, the time when H. vulgare and H. bulbosum
diverged (52). An alternative explanation is that the second gene
variant is the remainder of an earlier whole-genome duplication
that occurred 20 million years before the divergence of Oryza,
Brachypodium, and Hordeum from a common ancestor that existed
4147 million years ago (53). Because Oryza sativa and Brachyopodium distachyon encode only one CENH3 variant each, it is
possible that the second CENH3 variant in these grasses was lost.
Loss of a second CENH3 gene copy also occurred in Zea mays (47).
In maize, the allotetraploidization event probably occurred 11.4
million years ago (54). On the other hand, in other allopolyploid
organisms, such as Arabidopsis suecica (21), wild rice (55), or Nicotiana tabacum (56), multiple CENH3 types were identied.
Despite considerable protein sequence differences in the CATD
region, both CENH3 variants of H. vulgare are detectable in the
centromeric nucleosomes of all mitotic, meiotic, and extended
interphase chromosomes of barley. This observation suggests at
least two possibilities regarding the centromeric localization of
two CENH3 variants. Either both CENH3 variants are incorporated into the same nucleosomes forming heterodimers or barley
centromeres are composed of alternate blocks of CENH3- and
CENH3-containing nucleosomes. The centromeric composition
of alternating blocks of CENH3- and H3-containing nucleosomes
has previously been demonstrated for metazoan organisms (36, 57)
and plants (58). However, because of the limited resolution of light
microscopy, our immunostaining experiment on extended chromatin bers does not exclude the possible formation of CENH3and CENH3-containing heterodimers.
CENH3 Behavior in Stable Species Combinations. Our analysis demonstrates that cross-species incorporation of CENH3s occurs
PNAS | August 16, 2011 | vol. 108 | no. 33 | E501

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Fig. 6. Distribution of CENH3 and CENH3 in mitotic (A) and meiotic (B)
metaphase chromosomes and interphase nuclei of H. vulgare (C) as demonstrated by immunostaining. An overlap of CENH3 and CENH3 signals
was found for centromeres as well as for (D) up to 12-fold articially extended centromeres. (Scale bars: 10 m.)

of anti-grass CENH3 signals (Fig. S8A). Hence, both CENH3


variants of H. vulgare incorporate equally well into the centromeric nucleosomes of H. bulbosum.
Next, a stable H. vulgare-H. bulbosum 7H substitution line was
studied. Chromosome 7H of H. bulbosum does not encode any
CENH3 gene. The genotype of the H. vulgare-H. bulbosum 7H
substitution line was conrmed by in situ hybridization (Fig. S8C).
After immunostaining, for each CENH3 antibody combination,
up to 14 overlapping discrete signals per nucleus were detected
(Fig. S8B). Hence, CENH3 and CENH3 of H. vulgare can
functionally compensate for the missing CENH3 of H. bulbosum.
We then posed the question of whether barley centromeres still
specically incorporate HvCENH3 when barley chromosomes
carrying CENH3 genes are added to the genome of T. aestivum,
a species less closely related to H. vulgare than H. bulbosum. When
hybrids are formed, the centromere-specic histones of each parental species may operate in the context of a dual set of centromeric sequences. We used a wheat-barley double-disomic 1H + 6H
addition line. RT-PCR demonstrated (CENH3: primer pair 8/9,
CENH3: primer pair 3/10) the presence of both H. vulgare
CENH3 transcripts in a T. aestivum background (Fig. S8D). Finally,
double immunostaining on nuclei of the characterized wheatbarley addition lines with grass CENH3 (as a positive control), and
HvCENH3- or HvCENH3-specic antibodies was performed.
Anti-HvCENH3 and anti-grass CENH3 each detected up to 46
overlapping signals per nucleus (Fig. S8E), but no centromeric
incorporation of HvCENH3 was found in nuclei of wheatbarley 1H + 6H plants. Thus, no species-specic incorporation of
CENH3 occurs if CENH3 of both parents coexists in stable hybrids.
However, not all parental CENH3 variants necessarily undergo
centromere incorporation if multiple CENH3s coexist.

in stable species combinations, because both CENH3 and


CENH3 of H. vulgare were detected in all centromeres of stable
interspecic H. vulgare H. bulbosum plants. However, because
antibodies specic for CENH3s of H. bulbosum were not available, it was not possible to determine whether CENH3s of
H. bulbosum are incorporated into the centromeres of H. vulgare
H. bulbosum hybrids. A different situation was observed for
CENH3 and CENH3 of H. vulgare in wheat-barley 1H + 6H
addition lines. Despite transcription of both barley CENH3
variants, only HvCENH3-specic immunosignals were detected. Because CENH3s of wheat are probably more similar in
sequence to CENH3 of H. vulgare, HvCENH3 was preferentially incorporated into the centromeres of wheat and barley.
After sexual hybridization, the CENH3 of one parent can promote centromere functionality of the second parent despite centromere sequence differences. However, the CENH3 sequence
dissimilarity must not be too severe. This nding is strongly supported by the availability of a large number of chromosome addition lines. Even remotely related species, such as oat and maize,
can be sexually hybridized to produce fertile partial hybrids (59).
In this species combination, CENH3 of oat compensates for the
missing CENH3 of maize, because the maize CENH3 gene is silenced in the genetic background of oat in oat-maize chromosome
addition lines (16). Similarly, the CENH3 protein from A. thaliana
can be detected in the centromeres of all chromosomes of allotetraploid A. thaliana Arabidopsis arenosa hybrid A. suecica (21).
Our observation based on sexually generated hybrids supports
previous cross-species CENH3 incorporation experiments in
transgenic organisms (27, 51, 6062), for which it was reported
that CENH3s of closely related species can target centromeres in
alien species.
It will be interesting to study how many different CENH3
variants are incorporated into centromeres of polyploid species.
To what degree does the cross-capability between species depend on the ability of centromeres to incorporate different parental CENH3 variants? Does each CENH3 variant use its own
set of assembly factors in hybrids? However, in a transgenic
situation, CENH3 of a closely related species could get incorporated alone (62) or in combination with WT CENH3 (51,
60) with help from the loading machinery of the host organism.
Transgenic complementation of an inactive version of CENH3
was only possible if the heterologous CENH3 originated from
a closely related species (62). For instance GFP-CENH3 from
the close relative A. arenosa complemented A. thaliana cenh3-1,
which is consistent with the observation that A. thaliana CENH3
localizes to both A. thaliana and A. arenosa chromosomes in the
allopolyploid species A. suecica (21). However, GFP-CENH3
from a closely related Brassica species was targeted to centromeres but did not complement cenh3-1, indicating that kinetochore localization and centromere function of alien CENH3
can be uncoupled (62).
Centromeric Loss of CENH3 Protein Is Involved in the Process of
Uniparental Chromosome Elimination. CENH3 immunostaining of

H. vulgare H. bulbosum embryos demonstrated that uniparental centromere inactivation is, as previously postulated (11,
1619), a cause of mitosis-dependent chromosome elimination in
wide hybrids. Active centromeres of H. vulgare and H. bulbosum
were CENH3-positive, whereas inactive H. bulbosum centromeres were CENH3-negative or the amount of CENH3 was
reduced in unstable hybrids.
Elimination of H. bulbosum chromosomes is completed by 59
d after pollination (35). Because CENH3 is a stable protein (22),
sperm-derived centromere-incorporated CENH3 proteins are
likely to provide a residual kinetochore function of H. bulbosum
until it falls below a level critical for correct chromosome segregation, resulting in chromosome elimination. That a limited
fraction of parental H3 variants is transmitted to the progeny has
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been demonstrated for animal embryos (22). If preexisting


CENH3 is partitioned equally between duplicated sister centromeres and no de novo incorporation of CENH3 into H.
bulbosum centromeres occurs, its amount will be approximately
halved with each cell division. In humans, even 10% of the endogenous CENH3 can aid in efcient kinetochore assembly (63).
On the other hand, a reduced CENH3 amount can trigger centromere inactivity in neocentromeres of maize (64). Nevertheless, if a zygotic resetting of CENH3, as demonstrated for GFPtagged CENH3 in A. thaliana (65, 66), also exists in grasses,
active removal of both parental CENH3s before the rst zygote
division would occur and the reactivation of H. bulbosum centromeres in unstable hybrids would be diminished.
Despite transcription of all parental CENH3 genes in unstable
H. vulgare H. bulbosum hybrids and the existence of crossspecies incorporation of CENH3s in stable hybrids, the centromeres of H. bulbosum in unstable hybrids had a reduced
content of CENH3. Therefore, we assume that in unstable H.
vulgare H. bulbosum hybrids, no incorporation of CENH3 into
the centromeres of H. bulbosum takes place. The regulation of
CENH3 loading and assembly into centromeres is mediated by
a number of proteins, and the erroneous function of any of these
will result in a nonfunctional centromere (reviewed in 67). For
example, inactivation of the metazoan CENH3 interacting partner CENP-H, CENP-I, CENP-K, or CENP-N results in reduced
assembly of nascent CENH3 into centromeric chromatin and
causes defects in kinetochore assembly and chromosome congression (34, 68). However, with the exception of Mis12 of A.
thaliana (69), no other protein involved in the establishment and
maintenance of CENH3 chromatin has been identied in plants.
In unstable hybrids, we noted a different degree of condensation
between both parental chromosomes. The condensation process of
H. bulbosum chromosomes was often delayed (4). In agreement
with our observation, Bennett et al. (3) reported that H. bulbosum
requires more time to complete the cell cycle than H. vulgare.
Because the correct time of CENH3 deposition seems to be essential (25), cell cycle asynchrony (e.g., attributable to genotypic
differences) might interfere with the loading of H. bulbosum
nucleosomes with CENH3 in unstable hybrids. This, however, does
not exclude the possibility that other factors may result in the
failure to assemble active H. bulbosum centromeres in unstable
hybrids. Notably, in interspecic somatic hybrids of different
mammals, predominant loss of one parental genome occurs
(reviewed in 70). However, it is not known whether a comparable
process of chromosome elimination via loss of CENH3 is evolutionarily conserved and would also occur in animal hybrids.
The elimination process of chromosomes can be inuenced by
environmental conditions (8). A temperature above 18 C during
the early stages of hybrid embryo growth can promote chromosome elimination. How does temperature inuence the process
of chromosome elimination? Temperature-mediated changes in
nucleosome composition via altered deposition of histone variants by chaperons have recently been demonstrated for plants
(71). If temperature-mediated changes in centromeric nucleosome assembly occur, the temperature effect on the process of
uniparental chromosomes could be explained. However, it is not
known whether chaperones involved in CENH3 loading are
temperature sensitive.
On the basis of the above-mentioned observations, we propose
a possible model of how the mitosis-dependent process of uniparental chromosome elimination works in H. vulgare H. bulbosum
hybrid embryos (Fig. 7). After fertilization of the H. vulgare egg by
the H. bulbosum sperm, all parental CENH3 genes are transcriptionally active. Translation of HvCENH3s occurs, but whether
translation of HbCENH3s takes place is unknown. HvCENH3
is then loaded into the centromeres of H. vulgare but not of
H. bulbosum. Because of cell cycle asynchrony of the two parental
genomes, CENH3 incorporation probably only occurs in the
Sanei et al.

PNAS PLUS

than uniparental silencing of CENH3 genes is causing centromere inactivity.

SEE COMMENTARY

Materials and Methods


Plant Material, Crossing Procedure, and Preparation of Embryos. A series of
crosses were made between diploid H. vulgare Emir (female parent) and
diploid H. bulbosum lines Cb2920/4 and Cb3811/3 (pollen donors) (72). For H.
vulgare plants, two environments were used with different temperatures to
control chromosome elimination after pollination. A temperature higher than
18 C supports chromosome elimination, whereas a temperature lower than
18 C promotes retention of the parental chromosomes after pollination with
H. bulbosum (8). Barley spikes were emasculated 12 d before anthesis and
pollinated 1 d later with freshly collected H. bulbosum pollen. Embryo development was stimulated by spraying the spikes with an aqueous solution of
gibberellic acid at a rate of 75 ppm 1 d after pollination (details are provided
in SI Materials and Methods). For subsequent in situ hybridization or indirect
immunostaining, 6- to 13-d-old ovaries were isolated and xed in ethanol/
acetic acid (3:1) or 4% (wt/vol) paraformaldehyde in 1 microtubulestabilizing buffer (MTSB), respectively. Embryos were isolated under a stereomicroscope using ne needles.
Different wheat-barley addition lines as well as barley-H. bulbosum substitution lines were used for mapping of CENH3 coding genes (details are
provided in Fig. S4).

Fig. 7. Proposed model of how the mitosis-dependent process of uniparental


chromosome elimination operates in H. vulgare H. bulbosum hybrid embryos. (A) After fertilization of the H. vulgare egg by the H. bulbosum sperm,
all parental CENH3s are transcriptionally active. (B) Translation of HvCENH3s
occurs; whether translation of HbCENH3s occurs is not known. (C) Loading of
CENH3 (red) into the centromeres of H. vulgare but not of H. bulbosum occurs.
As a result of cell cycle asynchrony of the two parental genomes, CENH3 incorporation probably only occurs in the centromeres of H. vulgare during G2.
A reduced temperature during early embryogenesis promotes normal centromere activity of both parental genomes. (D) As a result of centromere inactivity in unstable hybrids, anaphase chromosomes of H. bulbosum lag
and subsequently form micronuclei. (E) Micronucleated H. bulbosum chromatin nally degrades, and a haploid H. vulgare embryo will develop.

centromeres of H. vulgare during G2. A low temperature during


early embryogenesis supports centromere activity of both parental
genomes. In unstable hybrids, H. bulbosum chromosomes lag because of centromere inactivity during anaphase, subsequently
forming micronuclei. Finally, micronucleated H. bulbosum chromatin will degrade, and a haploid H. vulgare embryo will develop.
Whether a comparable haploidization process takes place in A.
thaliana cenh3-1 null mutants expressing altered CENH3 proteins
that were crossed to WT plants remains to be studied (61).
In summary, we report four major conclusions regarding the role
of CENH3 in chromosomally stable and unstable interspecic
combinations (1). Diploid barley species encode two CENH3
variants whose gene products are intermingled throughout mitotic
and meiotic centromeres (2). In stable species combinations, crossspecies incorporation of CENH3 occurs despite centromeresequence differences. However, not all CENH3 variants get
incorporated into centromeres if multiple CENH3s are present in
species combinations (3). Centromere inactivity of H. bulbosum
chromosomes triggers the mitosis-dependent process of uniparental chromosome elimination in unstable H. vulgare H. bulbosum hybrids (4). Centromeric loss of CENH3 protein rather
Sanei et al.

FISH and Indirect Immunostaining. FISH and indirect immunostaining were


carried out as described by Gernand et al. (4) and Ma et al. (74), respectively.
FISH probes were labeled with Atto-590-dUTP or Atto-488-dUTP (Jena Bioscience) by nick translation. The following primary antibodies were used:
rabbit anti-RNA polymerase II CDC phospho Ser5 (catalog no. 39233; diluted
1:100; ACTIVE MOTIF), rabbit anti-histone H3K9me2 (catalog no. 07-441, diluted 1:300; Upstate), mouse anti-tubulin (catalog no. T 9026, diluted 1:100;
Sigma), and rabbit anti-grass CENH3 (40) (diluted 1:100), as well as HvCENH3and HvCENH3-specic antibodies (diluted 1:100). Epiuorescence signals
were recorded with a cooled CCD-camera (ORCA-ER; Hamamatsu). Imaging
was performed using an Olympus BX61 microscope and an ORCA-ER CCD
camera (Hamamatsu). Deconvolution microscopy was used for superior optical resolution of globular structures. All images were collected in gray scale
and pseudocolored with Adobe Photoshop 6 (Adobe). Projections (maximum
intensity) were done with the program AnalySIS (Soft Imaging System).
RNA Isolation, RT-PCR, and RACE PCR. RNA was extracted by either the TRIzol
method (75) or using the PicoPure RNA Isolation Kit (Arcturus). cDNA was
prepared from DNase I-treated RNA using the Reverse Aid H Minus First
Strand cDNA Synthesis Kit (Fermantas). PCR included the following steps:
94 C for 3 min, 40 cycles at 94 C for 1 min, annealing temperature for 1 min
and 10 s, and 72 C for 1 min and 30 s. Primers and annealing temperatures
are given in Table S1. For RACE PCR, the SMART RACE cDNA Amplication
Kit (Clontech Company) was used.
Analysis of PCR Fragments and Cleaved Amplied Polymorphic Sequence
Analysis. DNA fragments were ligated into the pGEM-T easy vector (Promega) and sequenced using the PGRC Sequencing Service (Leibnitz Institute of
Plant Genetics and Crop Plant Research). For cleaved amplied polymorphic
sequence analysis, puried PCR products were digested using AlwI or BanII,
size-fractionated by gel electrophoresis, and recorded.
Generation of H. vulgare CENH3-Specic Antibodies. Suitable CENH3 typespecic epitopes were identied [CQRRQETDGAGTSETPRRAGR and CAEGAPGEPTKRKPHRFR for generating HvCENH3-specic antibodies (a double-peptide
antibody) and CSKSEPQSQPKKKEKRAYR for generating HvCENH3-specic
antibodies]. The corresponding peptides were synthesized and used to immunize guinea pigs for generation of anti-HvCENH3 antibodies and rabbits for
generation of anti-HvCENH3 antibodies to analyze both CENH3 types simultaneously. Peptide synthesis, immunization of animals, and peptide afnity
purication of antisera were performed by Pineda (Antikrper-Service).
In Vitro Protein Synthesis and Western Blot Analysis. For in vitro protein
synthesis, the PURExpress cell-free transcription-translation system (New
England Biolabs) was used. Quantied protein samples were separated on

PNAS | August 16, 2011 | vol. 108 | no. 33 | E503

PLANT BIOLOGY

Isolation of Nuclei and Preparation of Stretched Chromatin Fibers. Nuclei were


isolated according to the method of Galbraith et al. (73). Extended chromatin
bers were prepared as described (39). The immunostaining procedure on the
stretched chromatin bers or on nuclei was the same as for squashed cells.

10% (wt/vol) SDS/PAGE gels using Tricin-SDS/PAGE running buffers (76),


blotted on nitrocellulose membranes, and then incubated rst with primary
antibodies (diluted 1:1,000) and then with secondary antibodies [anti-rabbit
IgG: IRDye800 conjugated, diluted 1:5,000 (LI-COR); anti-guinea pig IgG:
HRP-conjugated, diluted 1:5,000 (Dianova)].

Phylogenetic relationships were estimated by the neighbor joining


method (Lasergene).

Phylogenetic Analysis. Evolutionary analyses was conducted using deduced amino acid sequences of CENH3 in Z. mays, Saccharum ofcinarum,
O. sativa, A. thaliana, and and types of H. vulgare and H. bulbosum.

ACKNOWLEDGMENTS. We thank members of the Cytogenetics Department


of the Leibnitz Institute of Plant Genetics and Crop Plant Research for
helpful comments on the manuscript. Further, we thank Lu Ma for performing the FISH experiments and A. K. M. R. Islam (Australia) and T. R. Endo
(Japan) for providing the wheat-barley addition lines. This work was
supported by the Deutsche Forschungsgemeinschaft (Grant HO 1779/9-1)
and Marie Curie Actions (Grant 219313).

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PNAS | August 16, 2011 | vol. 108 | no. 33 | E505

Supporting Information
Sanei et al. 10.1073/pnas.1103190108
SI Materials and Methods
Plant Growth Conditions and Crossing Approaches. Two genotypes of

Hordeum bulbosum (Cb2920/4 and Cb3811/3) (1) were vegetatively propagated and vernalized for 78 wk at 4 C, with an 8-h
day length. Vegetative propagation is necessary because H. bulbosum is self-incompatible (2) and individual genotypes cannot
be established from seeds. After vernalization, the two genotypes
were maintained separately in cool glasshouses (temperatures
<18 C) with a 16-h day length.
For the H. vulgare (Emir) plants, two environments were used
with contrasting temperatures to control chromosome elimination
after pollination. One glasshouse was maintained with temperatures greater than 18 C for chromosome elimination, whereas
the other had temperatures less than 18 C to promote retention
1. Sanei M, et al. (2010) Interspecic hybrids of Hordeum marinum ssp. marinum x H.
bulbosum are mitotically stable and reveal no gross alterations in chromatin
properties. Cytogenet Genome Res 129:110116.

of the parental chromosomes after pollination with H. bulbosum.


Plants were cultivated until ear emergence in a cool glasshouse
and were then transferred to their respective environments.
Crossing was done conventionally by emasculating orets of the
female parent before anthesis; the spikes covered with bags to
prevent out-pollination and pollinated with freshly collected
pollen from the male parent. A ne spray of plant growth regulators was applied to orets 1 d (summer) or 1 and 2 d (winter)
after pollination to stimulate seed development and improve the
quality of the seeds. The mixture comprised 75 mg/L gibberellic
acid plus 1 mg/L dicamba, with or without 2 mg/L 2,4-dichlorophenoxyacetic acid. Twelve drops per liter of Tween 20 was
added as a surfactant. Immature embryos of various sizes were
excised under a stereomicroscope for further analysis.
2. Bothmer R, Salomon B, Linde-Laursen I (1995) Variation for crossability in a reciprocal,
interspecic cross involving Hordeum vulgare and H. lechleri. Euphytica 84:183187.

Fig. S1. Conrmation of anti-grass CENH3 cross-reactivity with CENH3s of H. bulbosum by indirect immunostaining. Immunostaining of metaphase (A) and
interphase (B) nuclei of H. bulbosum with anti-grass CENH3. (Scale bar: 10 m.)

Fig. S2. H. vulgare H. bulbosum hybrid anaphase cells show lagging chromosomes (arrowheads). (A), (B), and (C) show different examples. Immunostaining
with anti-grass CENH3 and anti-tubulin. (Scale bar: 10 m.)

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Fig. S3. Comparison of CENH3 and CENH3 of H. vulgare and of H. bulbosum with CENH3s of maize, rice, and sugar cane. Hb, H. bulbosum; Hv, H. vulgare;
Os, rice; So, sugar cane; Zm, maize. (A) Alignment of deduced amino acid sequences. The CENH3-typical N-helix, 1-helix, 2-helix, 3-helix, loop 1 region, and
CAT domain are indicated. (B) Phylogenetic analysis shows that the CENH3s form a distinct subcluster with the CENH3s of maize, rice, and sugar cane. CENH3s
of Hordeum species form a separate cluster. CENH3 of A. thaliana was used as an outgroup. At, A. thaliana.

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Fig. S4. Chromosome mapping of CENH3s and CENH3s in H. vulgare (A and B) and in H. bulbosum (C and D). Mapping of CENH3s (A) and CENH3s (B) of H.
vulgare using wheat-barley addition lines. The bands corresponding to the barley CENH3s are indicated by arrowheads. PCR was performed on the genomic DNA
samples of (from left to right) wheat (Chinese Spring), barley (Betzes), and seven barley (Betzes) chromosome addition lines in wheat (Chinese Spring) (1, 2).
Because the 1H addition line causes severe sterility, double-disomic addition 1H and 6H was used. For amplication of HvCENH3 and HvCENH3, the primer pairs
8/9 and 3/10 were used, respectively. Chromosome mapping of CENH3s (C) and CENH3s (D) of H. bulbosum using H. vulgare lines with substituted H. bulbosum
chromosomes. (C) Amplication of CENH3 in H. bulbosum but not in the substitution lines (2H7H) (3, 4), except for 1H, indirectly indicates that CENH3 is
located on 1H of H. bulbosum A substitution with 1H of H. bulbosum is not available. (D) Amplication of the chromosomal location of HbCENH3 using the 6H
H. bulbosum substitution line in the barley genome; the result shows that this gene is located on chromosome 6 of H. bulbosum. For amplication of HbCENH3
and HbCENH3, the primer pairs 4/5 and 3/11 were used, respectively. Amplication of GAPDH with primers 12 and 13 was used as a positive control.

1.
2.
3.
4.

Islam AKMR, Shepherd KW (2000) Isolation of a fertile wheat-barley addition line carrying the entire barley chromosome 1H. Euphytica 111:145149.
Islam AKMR, Shepherd KW, Sparrow DHB (1981) Isolation and characterization of euplasmic wheat-barley chromosome addition lines. Heredity 46:161174.
Pickering RA (1992) Monosomic and double monosomic substitutions of Hordeum bulbosum L. chromosomes into H. vulgare L. Theor Appl Genet 84:466472.
Thiele V, Pickering RA, Melz G, Pohler W (1992) Identication of Hordeum bulbosum chromosomes in H. vulgareH. bulbosum substitutions using isozyme markers. Genome 35:
454460.

Fig. S5. Transcriptional analysis of CENH3 (A) and CENH3 (B) of H. vulgare and H. bulbosum in stable and unstable plants 5 and 7 d after pollination (DAP) of
old H. vulgare H. bulbosum hybrid embryos by RT-PCR. Only H. bulbosum-derived CENH3 and CENH3 transcripts were digestible with AlwI and BanII,
respectively. As a control, RT-PCR products of H. vulgare and H. bulbosum plants were used. DAP, days after pollination; H.b., H. bulbosum; H.v., H. vulgare.

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Fig. S6. Distribution of CENH3 and CENH3 at different stages of mitosis (A) and meiosis (B) in H. vulgare is demonstrated by immunostaining. (Scale bars: 10 m.)

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Fig. S7. Conrmation of anti-HvCENH3 and anti-HvCENH3 species specicity by indirect immunostaining of H. bulbosum and T. aestivum chromosomes.
Anti-grass CENH3 was used as a positive control. (Scale bar: 10 m.)

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Fig. S8. Cross-species incorporation analysis of CENH3 and CENH3 of H. vulgare in a closely related species. All centromeres of stable H. vulgare H.
bulbosum hybrid nuclei (A) and of an H. vulgare-H. bulbosum 7H substitution incorporating CENH3 and CENH3 derived from H. vulgare (B) are shown. (C)
Genotype conrmation of an H. vulgare-H. bulbosum 7H substitution line by in situ hybridization. Mitotic H. vulgare-H. bulbosum 7H substitution chromosomes after FISH using genomic H. bulbosum DNA (green); (AGGGAG)n, a barley-specic centromere repeat (yellow); and 5S rDNA (red) as probes. Circles
Legend continued on following page

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indicate chromosome 7H of H. bulbosum (D) Transcription and cross-species incorporation analysis of CENH3 and CENH3 of H. vulgare in a wheat-barley
double-disomic 1H + 6H addition line. RT-PCR demonstrates transcription of both CENH3 variants of barley in the addition line. (E) All barley and wheat
centromeres incorporate CENH3 (Upper) but not CENH3 (Lower) of H. vulgare despite transcription. Anti-grass CENH3 was used as an internal control. (Scale
bar: 10 m.)

Table S1.

List of primers

No.

Name of primer

Sequence 53

Annealing
temperature, C

1
2
3
4
5
6
7
8
9
10
11
12
13

Degenerate-F
Degenerate-R
Race-CENH3-F
Hv + Hb-F
Hv + Hb-R
CENH3-F
CENH3-3-R
CENH3-F
CENH3-R
CENH3-R
HbCENH3-R
GAPDH-F
GAPDH-R

GTRGCRCTGCGGGAGATCAGGA
CTBGCRAGYTGYATGTCCTTTT
GTGGCCACTGCGGGAGATCAGGAAGTACC
CGGGCACGTCCGAGACTCC
GTAGAATTCGGTGACCTCCTTGACC
ATGGCTCGCACGAAGAAAACGG
GCAAAGGCCGAGAAGTCAGATG
AGAAGAAGATCGGGTCCGCTA
GTGCAAACGGGATGAGAAAATT
GTCGGCTTGCTCTCCTTCTTGTTCG
ATGGCGTCGGCTTGTTGGACCC
CAATGATAGCTGCACCACCAACTG
CTAGCTGCCCTTCCACCTCTCCA

68
61
72
69
66
64.5
64.5
64.5
59
68
68
59
59

F, forward; R, reverse.

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